Investigations of the intracellular signals that direct a multipotent hematopoietic stem cell to establish the erythroid lineage during ontogeny have successfully focused on the transcription factors that generate the erythroid program. Nevertheless, how these important factors are themselves induced and regulated, and how this relates to the extracellular molecules known to stimulate red cell production, remain a mystery. We have focused on the regulation of the EKLF (erythroid Kruppel- like factor) gene as a means to fill in these gaps in knowledge. EKLF is an erythroid cell-specific transcription factor whose biological properties and genetic expression profile make it eminently suitable for such analyses. This renewal builds upon our previous studies, which have determined the chromosomal structure, sequence conservation, minimal in vivo enhancer element, and in vitro protein-DNA interactions of the EKLF transcription unit. We have also achieved significant purification of one of the DNA binding activities, and have established serum-free conditions whereby the dependence on soluble growth factors of EKLF expression during embryonic stem cell differentiation can be monitored. We thus propose to illuminate how production of an erythroid cell-specific intracellular regulatory molecule is accomplished and is related to extracellular signals by: (1) Identifying the protein(s) that interact with the EKLF enhancer element; (2) Testing the functional importance of cis-acting elements, previously characterized only in transfection assays, in transgenic mice and in differentiating embryoid stem cells; (3) Identifying the extracellular molecules that induce EKLF expression by means of a novel assay that takes advantage of the ability to differentiate embryonic stem cells into embryoid bodies in the absence of serum. Establishing the controls involved in EKLF expression will likely focus attention on a subset of molecules involved in auto-or cross-regulation of primary erythroid factors and their role in signal transduction. These studies are therefore relevant to delineating the infra- and extra-cellular mechanisms that regulate commitment of hematopoietic stem cells during early mammalian development, and in addition have clinical relevance for stem cell-based therapies that depend on expansion of erythroid progenitor cells for their use in transplantation.